Long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination.

Canine parvovirus (CPV) modified live virus vaccines are able to infect vaccinated dogs replicating in the bloodstream and enteric mucosa. However, the exact duration and extent of CPV vaccine-induced viremia and fecal shedding are not known. With the aim to fill this gap, 26 dogs were administered two commercial vaccines containing a CPV-2 or CPV-2b strain and monitored for 28 days after vaccination. By using real-time PCR, vaccine-induced viremia and shedding were found to be long lasting for both vaccinal strains. Vaccinal CPV-2b shedding was detected for a shorter period than CPV-2 (12 against 19 mean days) but with greater viral loads, whereas viremia occurred for a longer period (22 against 19 mean days) and with higher titers for CPV-2b. Seroconversion appeared as early as 7 and 14 days post-vaccination for CPV-2b and CPV-2 vaccines, respectively. With no vaccine there was any diagnostic interference using in-clinic or hemagglutination test, since positive results were obtained only by fecal real-time PCR testing. The present study adds new insights into the CPV vaccine persistence in the organism and possible interference with diagnostic tests.

Introduction

Along with canine coronavirus [1], [2], canine parvovirus (CPV) is the main cause of acute hemorrhagic enteritis in young dogs, which may display very severe clinical signs such as leukopenia, fever, inappetence, hemorragic diarrhea, dehydration and death [3]. Currently, three different antigenic variants are known, namely CPV-2a, CPV-2b and CPV-2c, which are variously distributed worldwide [4], [5], [6], [7], [8]. The original type CPV-2, albeit no longer circulating in the field, is still contained in most CPV vaccines, whereas few commercially-available vaccines are prepared with CPV-2b [3]. Although CPV-2c is becoming the predominant strain in several countries [4], [5], [6], [7], [8], to date there are no licensed vaccines that contain the newest variant. Both CPV-2 and CPV-2b vaccinal strains cause viremia and are shed with the feces of immunized dogs, as also shown by their detection in specimens from dogs displaying acute gastroenteritis shortly after vaccination, alone or in addition to CPV field strains or other pathogens [9]. However, nothing is known about the real duration and extent of viremia and shedding of vaccinal CPVs.

The aim of the present paper is to report the results of virological investigations carried out on pups routinely undergoing CPV vaccination in order to assess the vaccine virus viremia and shedding with the feces.

Materials and methods

Vaccines

Two commercial modified live virus (MLV) vaccines were used in the study, Nobivac® PUPPY CP (Intervet Italia S.r.l., Milan, Italy) and Duramune® DAPPI + LC (Zoetis Italia S.r.l., Rome, Italy), containing >107 and 104.7–106.5 tissue culture infectious doses of CPV-2 strain 154 and CPV-2b strain SAH, respectively.

Vaccination protocol

A total of 26 pups belonging to two different breeding kennels were recruited in the study with the written consent of the breeders. The dogs, 16 males and 10 females, consisted of 13 American cocker spaniels and 13 Labrador retrievers that were randomly divided into two vaccine groups (on the basis of the vaccine administered) in order to include both breeds and genders in each group. At the age of 6 weeks, all pups were bled to collect sera that were submitted to hemagglutination inhibition (HI) in order to predict the most appropriate age of vaccination on the basis of their maternally-derived antibody (MDA) titers [10]. When MDA titers were below the levels interfering with CPV vaccination (<1:20), pups were administered subcutaneously one dose of CPV-2 or CPV-2b vaccine according to the vaccine group.

Sample collection

Vaccinated animals were monitored for a period of 28 days post-vaccination (dpv) in order to evaluate vaccinal strain viremia, shedding and seroconversion.

For virological investigations, EDTA–blood samples and fecal specimens were collected from vaccinated pups at dpv 3, 7, 10, 14, 17, 21, 24 and 28 by means of jugular venipuncture and anal swabs, respectively. Serological testing was carried out on serum samples taken once a week from each dog.

Virological investigations

In-clinic test

The in-clinic test was carried out with the commercial kit WITNESS Parvo test (Synbiotics Corporation, Pfizer), as previously described [11]. The fecal material was collected on the test kit swab following the manufacturer's instructions. The extraction buffer/conjugate was dispensed into the sample tube via the kit swab. Then, the sample swab was inserted into the tube containing the liquid and vortexed. The extracted fecal material/conjugate liquid was transferred to the WITNESS Parvo device using the swab pipette for the test kit as per manufacturer's instructions.

Hemagglutination (HA)

Two-fold dilutions of the supernatant of each fecal homogenate were made in PBS (pH 7.2) starting from a 1:2 dilution [12]. Tests were carried out in 96-well V-plates (50 μl of sample dilution per well); equal amounts of a suspension containing 0.8% pig erythrocytes and 1% fetal calf serum (FCS) were added to each dilution. Results were read after 4 h at +4 °C and expressed as the reciprocal of the highest sample dilutions able to produce HA.

Real-time PCR assays for CPV detection, quantification and characterization

Specimens were homogenized (10%, w/v) in Dulbecco's modified Eagle's medium (DMEM) and subsequently clarified by centrifuging at 2500 ×  g for 10 min. Viral DNA was extracted from the supernatants of fecal homogenates by boiling for 10 min and chilling on ice. To reduce residual inhibitors of DNA polymerase activity to ineffective concentrations, the DNA extract was diluted 1:10 in distilled water [13].

Detection and quantification of CPV DNA was obtained by real-time PCR using a conventional TaqMan probe [13]. Briefly, the 25-μl reaction contained 12.5 μl of master mix (Bio-Rad Laboratories S.r.l., Milan, Italy), 600 nM of primers (5′-AAACAGGAATTAACTATACTAATATATTTA-3′) and CPV-Rev (5′-AAATTTGACCATTTGGATAAACT-3′), 200 nM of probe CPV-Pb (5′-TGGTCCTTTAACTGCATTAAATAATGTACC-3′) and 10 μl of standard or template DNA. For the standard-curve construction, ten-fold dilutions of a plasmid containing the nearly full-length CPV genome (kindly supplied by C.R. Parrish, Cornell University, Ithaca, NY, USA) were processed. All standard dilutions and unknown samples were tested in duplicate. The following thermal protocol was used: activation of iTaq DNA polymerase at 95 °C for 10 min and 40 cycles consisting of denaturation at 95 °C for 15 s, primer annealing at 52 °C for 30 s and extension at 60 °C for 1 min.

A panel of minor groove binder (MGB) probe assays able to predict the viral type [14], [15] and to discriminate between vaccine and field strains of CPV [16], [17] was used to confirm the viral strain detected in vaccinated dogs.

Serological investigations

Sera of dogs collected at dpv 0, 7, 14, 21 and 28 were submitted to an HI test using a standardized protocol [10]. The tests were performed at +4 °C in 96-well V-plates, using 10 hemagglutinating units of CPV-2 antigen and 0.8% pig erythrocytes. Two-fold dilutions in PBS of each serum sample, starting from 1:10, were tested. The HI titer was indicated as the highest serum dilution completely inhibiting viral hemagglutination.

Statistical analysis

The data were analyzed using the Prism 6 software (version 6.0d). All hypothesis tests were conducted at the 0.05 level of significance (two-sides). The areas under curve (AUC) for the viremia and fecal shedding of the vaccine viruses were calculated for each group and the statistical significance was evaluated using the Wilcoxon–Mann–Whitney test. Prior to analysis the AUC values were logarithmically transformed. HI antibody titers were also analyzed by the Wilcoxon test.

Results

CPV post-vaccinal viremia

By real-time PCR, dogs immunized with the CPV-2 strain displayed viremia for 19 mean days, from dpv 3 to 21, with mean viral titers peaking at dpv 10 (1.12 × 106 DNA copies 10 μl−1 of template). In dogs vaccinated with CPV-2b, viremia started at dpv 3 and stopped at dpv 24 (22 mean days), with maximal titers of 6.39 × 107 DNA copies 10 μl−1 of template being observed at dpv 7 (Fig. 1A).

Fig. 1

CPV viremia (A), fecal shedding (B) and seronversion (C) in dogs administered CPV-2 or CPV-2b vaccines. CPV viremia (A) and fecal shedding (B) are expressed as mean viral DNA copy numbers 10 μl−1 of template, while antibody responses (C) are presented as log2 geometric means of HI titers. Error bars indicate the calculated standard errors.

Minor groove binder (MGB) probe assays developed to predict the antigenic type and discriminate between filed and vaccine strains [14], [15], [16], [17] confirmed the positivity for the vaccine strain (CPV-2 or CPV-2b) according to the vaccine group.

The mean values for the area under the curve of real time PCR results from EDTA–blood for the observation period were 4.55 × 106 and 2.37 × 108 for CPV-2 and CPV-2b vaccinated dogs, respectively (P  < 0.001).

CPV post-vaccinal shedding

None fecal swab collected after vaccination tested positive by either in-clinic testing or HA. By real-time PCR, CPV-2 immunized pups shed the virus with the same pattern observed for viremia (19 mean days), although viral DNA loads detected in the feces were slightly lower than those observed in the blood. The highest DNA loads (mean of 3.80 × 105 DNA copies 10 μl−1 of template) were shed at dpv 7. Vaccinal CPV-2b shedding occurred for a shorter period (12 mean days, from dpv 3 to 14) but with greater viral load, which peaked at dpv 10 (mean titer of 1.12 × 106 DNA copies 10 μl−1 of template) (Fig. 1B). The vaccine strain shed through the feces of CPV-2 or CPV-2b vaccinated dogs was confirmed by MGB probe testing [14], [15], [16], [17].

The area under curve that resulted from the real-time PCR titers detected in fecal specimens during the entire observation period displayed mean values of 1.61 × 106 and 4.04 × 106 for pups immunized against CPV-2 and CPV-2b, respectively (P  < 0.05).

CPV seroconversion

Fig. 1C illustrates the antibody patterns observed in the two vaccine groups. In dogs administered the CPV-2 vaccine, HI antibodies against CPV were first detected at dpv 14 (geometric mean titers of 8.10) and reached maximal values at dpv 28 (geometric mean titers of 10.10). In contrast, CPV-2b vaccinated dogs seroconverted already at dpv 7 (geometric mean titers of 1.97), although similar to the other vaccine group, maximal titers were reached at dpv 28 (geometric mean titers of 12.63). HI antibody titers induced by CPV-2b were generally higher in comparison to those raised against CPV-2 (P  < 0.001).

Discussion

International guidelines for dog vaccination recommend the use of MLV vaccines to prevent infection by viruses included in the core vaccines, i.e. CPV, canine distemper virus and canine adenovirus [18]. MLV CPV strains are able to replicate in the bloodstream and intestinal mucosa of vaccinated dogs, despite the unnatural route of administration (intramuscular or subcutaneous instead of oronasal), causing viremia and fecal shedding [19], [20]. In such a circumstance, the detection of CPV or its nucleic acid in the feces of vaccinated dogs could provide false-positive results, leading to a misdiagnosis of the disease probably caused by other enteric pathogens of dogs, i.e., canine coronavirus, canine distemper virus, reoviruses, rotaviruses, Salmonella spp., protozoa, parasites, etc. Moreover, there is the need to rule out vaccine-induced disease due to regaining of virulence of the vaccine virus. In a recent study [9], molecular testing of 29 fecal samples collected from dogs with diarrhea following CPV vaccination showed that in most cases those animals were infected by CPV field strains or other canine pathogens. However, 11 dogs were found to shed CPV vaccine strains in combination with field strains or other agents, whereas three samples tested positive for the vaccine strain without evidence of canine pathogens. To date, there are no molecular studies investigating the persistence of CPV vaccinal strains in the canine organism.

In order to assess the exact duration and extent of CPV vaccine-induced viremia and fecal shedding, 26 dogs were administered two commercial vaccines containing a CPV-2 or CPV-2b strain and monitored for 28 days after vaccination. By using real-time PCR, vaccine-induced viremia and fecal shedding occurred at higher titers for CPV-2b than for CPV-2. Interestingly, while viremia was observed for more days for CPV-2b (22 against 19 means days), the old-type vaccine strain was shed in the feces for a longer period (19 mean days) than the CPV-2b virus (12 mean days). Viral DNA loads detected by real-time PCR in the blood and feces of immunized dogs were generally much lower than those observed at least for the CPV variants causing natural or experimental infections [10], [21], [22]. In the feces, the field viruses can reach loads above 1012 DNA copies 10 μl−1 of template, while vaccinal strains were shed at maximal titers of 105–106 DNA copies 10 μl−1 of template (mean values). As for the duration of the fecal shedding, it was surprising that CPV-2b, albeit shed at higher titers, was detected in the feces for a shorter period in comparison to the CPV-2 vaccinal strain. As expected, none vaccinated dogs displayed any clinical signs after vaccination. Moreover, the impact of concurrent infections on the MLV persistence was not investigated, thus requiring further studies.

Another striking finding was that both the in-clinic (ELISA-based) and laboratory (HA) assays that are routinely employed for CPV diagnosis gave negative results on the fecal samples from vaccinated dogs, even when the virus reached discrete titers that should be detected by the tests [12], [23], [11]. In-clinic testing and HA were proved to be poorly sensitive compared with nucleic acid-based assays, mainly due to sequestration of viral particles by gut antibodies especially during late infection [12], [23], [11]. In the present study, the maximal CPV DNA titers (more than 105 and 106 mean DNA copies 10 μl−1 of template for CPV-2 and CPV-2b viruses) were detected as early as one week post-vaccination when serum HI antibody titers were still absent or very low (Fig. 1C). However, the kinetic of the mucosal antibody response was not investigated in vaccinated animals, so that the presence of high titers of gut antibodies, likely mucosal IgAs, could not be ruled out. Our findings are in contrast with those of a recent study aiming to evaluate the feline panleukopenia virus (FPLV) vaccine-induced interference with fecal parvovirus diagnostic testing in cats [24]. This study showed that some cats had in-clinic assay positive results after FPLV vaccination although all kits employed contained CPV antibodies.

In conclusion, the present study adds new insights into the CPV vaccine persistence in the organism and possible interference with diagnostic tests.

Conflict of interest

The authors declare no conflict of interest.